The Ups and Downs of Genome Size Evolution in Polyploid Species
of Nicotiana (Solanaceae)
I. J. LEITCH1,*, L. HANSON1, K. Y. LIM2, A. KOVARIK3, M. W. CHASE1, J. J. CLARKSON1
and A. R. LEITCH2
1Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK,2School of Biological and Chemical
Sciences, Queen Mary, University of London, London E1 4NS, UK and3Institute of Biophysics, Academy of Sciences
of the Czech Republic, CZ-61265 Brno, Czech Republic
Received: 11 July 2007Returned for revision: 2 October 2007 Accepted: 5 December 2007Published electronically: 24 January 2008
†Background In studies looking at individual polyploid species, the most common patterns of genomic change are
that either genome size in the polyploid is additive (i.e. the sum of parental genome donors) or there is evidence of
genome downsizing. Reports showing an increase in genome size are rare. In a large-scale analysis of 3008 species,
genome downsizing was shown to be a widespread biological response to polyploidy. Polyploidy in the genus
Nicotiana (Solanaceae) is common with approx. 40 % of the approx. 75 species being allotetraploid. Recent
advances in understanding phylogenetic relationships of Nicotiana species and dating polyploid formation enable
a temporal dimension to be added to the analysis of genome size evolution in these polyploids.
†Methods Genome sizes were measured in 18 species of Nicotiana (nine diploids and nine polyploids) ranging in
age from ,200 000 years to approx. 4.5 Myr old, to determine the direction and extent of genome size change fol-
lowing polyploidy. These data were combined with data from genomic in situ hybridization and increasing amounts
of information on sequence composition in Nicotiana to provide insights into the molecular basis of genome size
†Key Results and Conclusions By comparing the expected genome size of the polyploid (based on summing the
genome size of species identified as either a parent or most closely related to the diploid progenitors) with the
observed genome size, four polyploids showed genome downsizing and five showed increases. There was no dis-
cernable pattern in the direction of genome size change with age of polyploids, although with increasing age the
amount of genome size change increased. In older polyploids (approx. 4.5 million years old) the increase in
genome size was associated with loss of detectable genomic in situ hybridization signal, whereas some hybridization
signal was still detected in species exhibiting genome downsizing. The possible significance of these results is
Key words: Genome downsizing, genome size, Nicotiana, polyploidy, sequence elimination, Solanaceae.
The genus Nicotiana, comprising approx. 75 species,
displays a range of genomic changes including gene con-
version, tandem and dispersed sequence evolution, interge-
nomic translocations, dysploidy, polyploidy, etc. (Kenton
et al., 1993; Matzke et al., 2004; Melayah et al., 2004;
Dadejova et al., 2007; Lim et al., 2007; Kovarik et al.,
2008). Given the increasingly robust phylogenetic frame-
work now available (Chase et al., 2003; Clarkson et al.,
2004, 2005), Nicotiana is thus ideally suited to study pat-
terns of genome evolution.
Polyploidy is common in the genus, with approx. 40% of
species being allotetraploid. They comprise (a) N. tabacum
(section Nicotianae), (b) N. rustica (section Rusticae),
(c) N. arentsii (section Undulatae), (d) N. clevelandii and
N. quadrivalvis (section Polydicliae), (e) N. nudicaulis,
N. repanda, N. nesophila and N. stocktonii (section
Repandae) and (f ) all approx. 23 species in section
Suaveolentes. All polyploids, except some of those in
section Suaveolentes, are 2n ¼ 4x ¼ 48, representing a dou-
bling of the diploid chromosome number for the genus
(2n ¼ 2x ¼ 24).InsectionSuaveolentes, polyploid
evolution has been accompanied by changes in chromosome
number, probably through dysploid reductions via chromo-
some deletions or fusions (2n ranges from 32 to 48).
Based on cytological and floral morphology, combined
with sequence data from plastid and nuclear genes
(Goodspeed, 1954; Aoki and Ito, 2000; Chase et al.,
2003; Clarkson et al., 2004), the likely parentage of
nearly all allotetraploid species has now been determined,
together with estimates of their ages based on combining
molecular clock analysis and calibration with the ages of
oceanic volcanic islands (Clarkson et al., 2005; Kovarik
et al., 2008) (Fig. 1). These data show that the polyploid
(N. tabacum, N. rustica and N. arentsii) are each estimated
to have arisen ,200 000 years ago, followed by the two
species in section Polydicliae,
1 million years (Myr) old, and the five species in section
Repandae, which are approx. 4.5 Myr old (Clarkson
et al., 2005). Theoldest
Suaveolentes, are considered to have originated from a
single polyploid event .10 Myr ago, followed by specia-
tion to produce the approx. 23 species known today.
Nicotiana polyploids not only vary in age but also in the
relatedness of the parental species contributing genomes to
inage. The youngest
polyploids, in section
* For correspondence. E-mail I.Leitch@kew.org
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the polyploid nucleus. For example, N. arentsii is an
intrasectional polyploid, the hybrids and diploid progenitors
all belonging to section Undulatae. In N. rustica (section
Rusticae) the parental species are in closely related sections
(Paniculatae and Undulatae) (Clarkson et al., 2004;
Kovarik et al., 2008). In contrast, the diploid species
giving rise to N. tabacum and polyploids in section
Repandae, Polydicliae and Suaveolentes are from distantly
related sections (Figs 1 and 2).
Insights into contrasting patterns of molecular evolution
in Nicotiana polyploids have been gained through the
study of natural and synthetic species. These have involved
analysing both specific DNA sequences [e.g. ribosomal
DNA (rDNA), non-coding tandem repeats and retrotranspo-
sons] and global genome organization using fluorescent in
situ hybridization (FISH) [including genomic in situ
hybridization (GISH); Kovarik et al., 1996, 2004; Chase
et al., 2003; Lim et al., 2004, 2005, 2006b, 2007;
Clarkson et al., 2005; Skalicka et al., 2005; Petit et al.,
2007]. Such studies have shown that allopolyploidy in
Nicotiana has been accompanied by numerous genetic
changes including (depending on the polyploid species in
(Matyasek et al., 2003; Kovarik et al., 2004; Clarkson
et al., 2005), intergenomic translocations (Kenton et al.,
1993; Chase et al., 2003; Lim et al., 2004) and changes
in copy number and organization of both tandem and dis-
persed repeats (Melayah et al., 2004; Petit et al., 2007).
By combining these data with the known ages of the poly-
ploids, a temporal perspective on polyploidy evolution has
been obtained. Lim et al. (2007) showed that during early
evolution of Nicotiana polyploids (i.e. those ,200 000
FIG. 1. Summary of phylogenetic relationships of Nicotiana species with proposed origins of polyploids. Data used in analyses include plastid and
internal transcribed spacer loci. Figure modified and adapted from Knapp et al. (2004) using more recent phylogenetic information taken from glutamine
synthase (J. J. Clarkson et al., unpubl. res.). Uncertainty concerning one of the parental genome donors for sections Polydicliae and Suaveolentes is
indicated by question marks.
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana806
FIG. 2. Direction of genome size evolution in Nicotiana polyploids varying in age from ,200000 years (A–C), approx. 1 Myr (D) to approx. 4.5 Myr
(E) old. The observed versus expected 1C DNA amounts in nine polyploids were based on comparison between genome sizes determined for diploid and
polyploid species using genome size data taken from Table 1. Putative diploid parental genome donors used for comparison of the various polyploids are
based on a range of molecular and cytogenetic results (see text).
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana 807
years old) intergenomic translocations and rearrangement
and loss of repeated DNA sequences may take place.
Over the next 1–2 Myr, considerable exchange of repeats
between the parental genomes becomes apparent, and by
5 Myr there can be near complete genomic turnover includ-
ing evolution of new repeats not present in the parental
Studies of genome size evolution in polyploids in a
diverse range of angiosperms have been conducted at a
number of levels. For specific polyploid species, there are
many reports showing no change or a decrease in DNA
amount relative to the proposed progenitor species (see
review in Leitch and Bennett, 2004); those showing an
increase are much rarer but include a few naturally occur-
ring species of Hordeum (Jakob et al., 2004) and artificially
induced dihaploids of Nicotiana (Dhillon et al., 1983). At
the other end of the spectrum, large-scale analyses combin-
ing available genome size data for 3008 angiosperms have
led to the proposal that genome downsizing is a widespread
biological response to polyploidization leading to diploidi-
zation of the polyploid genome (Leitch and Bennett, 2004).
Such conclusions have been supported by comparative mol-
ecular studies. For example, nearly 80% of the duplicated
genes have been lost from rice (Oryza sativa) since the
polyploid event that gave rise to it approx. 70 Myr ago
(Wang et al., 2005), and .50 % of the duplicated genes
in maize (Zea mays) have been lost since its polyploid
origin approx. 3–11 Myr ago (Messing et al., 2004). In
addition, comparative analyses of DNA sequences sur-
rounding the AdhA locus in diploid and polyploid
Gossypium genomes suggest that polyploidization may
lead to increased rates of illegitimate recombination result-
ing in a greater loss of DNA from polyploids than related
diploids (Grover et al., 2007b). Rapid loss of DNA (based
both on genome size measurements or loss of AFLP and/
or RFLP bands or specific sequences) has also been
reported following synthesis of various artificial hybrids
and polyploids of Brassica (Song et al., 1995), Nicotiana
(Skalicka et al., 2003, 2005; Petit et al., 2007) and
Aegilops and Triticum (Ozkan et al., 2001, 2003;
Kashkush et al., 2002; Levy and Feldman, 2004).
Given contrasting ages of Nicotiana polyploids (,200000
years to .10 Myr), differences in the extent of genome
divergence in diploid progenitors and the diversity of mol-
ecular changes that have been documented to occur follow-
ing polyploidization, it seems timely to examine patterns of
TABLE 1. List of Nicotiana species studied together with chromosome number, 1C and 4C DNA amount, calibration standard
and method of estimating genome size
(where available) and
source of material*2n
4C DNA (+ s.d.)
1C DNA (+ s.e.)
N. tabacum L.
N. attenuata Torrey ex S.Watson
N. knightiana Goodsp.
N. paniculata L.
N. clevelandii A.Gray
N. quadrivalvis Pursh.
N. repanda Willd.
N. nesophila I.M.Johnston
N. nudicaulis S.Watson
N. stocktonii Brandegee
N. rustica L.
N. sylvestris Speg. & Comes
N. tomentosiformis Goodsp.
N. obtusifolia M.Martens & Galeotti
N. arentsii Goodsp.
N. glutinosa L.
N. undulata Ruiz & Pav.
N. wigandioides Koch & Fintelm.
Nee et al. 51789a
48 20.70 (1.34)5.2 (0.060) PisumFe
24 9.93 (0.05)2.5 (0.002)Pisum FC
48 21.19 (0.08) 5.3 (0.040)PisumFC
24 10.78 (0.10) 2.7 (0.002)Pisum FC
Nee et al. 51771a
2410.97 (0.19)2.7 (0.020) PisumFC
246.18 (0.03)1.5 (0.002)SolanumFC
Nee et al. 51764a
* Source of material: a ¼ New York Botanic Garden, New York, USA; b ¼ USDA, North Carolina State University, Raleigh, NC, USA; c ¼ IPK,
Gatersleben, Germany; d ¼ Botanical and Experimental Garden, Radboud University Nijmegen,The Netherlands; e ¼ Chelsea Physic Garden, London,
UK; f ¼ Royal Botanic Gardens, Kew, UK.
†Species and 4C values used for calibration standards are as follows: Pisum ¼ Pisum sativum cv. Minerva Maple, 4C ¼ 17.52 pg; Solanum ¼
Solanum lycopersicum, 4C ¼ 4.00 pg; Hordeum ¼ Hordeum vulgare cv. Sultan, 4C ¼ 22.24 pg; Zea ¼ Zea mays CE-777, 4C ¼ 11.34 pg.
‡FC ¼ flow cytometry; Fe ¼ Feulgen microdensitometry.
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana808
genome size evolution in Nicotiana polyploids of different
ages. Although there have been several previous studies
reporting genome sizes for species of Nicotiana, the most
extensive has been by Narayan (1987) who listed
C-values for 51 Nicotiana taxa, including all but two of
the species studied here. However, in his discussion,
genome size evolution was only assessed in the three
N. arentsii), and there were considerable discrepancies
between some of his genome size estimates compared
with those previously published by himself (Narayan and
Rees, 1974), other authors (e.g. Ingle et al., 1975;
Galbraith et al., 1983) and here (see Discussion). Thus,
genome sizes were estimated in nine diploid and nine poly-
ploid species. Unfortunately, taxonomic relationships and
parental genome donors of polyploid Suaveolentes species
are not yet sufficiently understood to be investigated in
METHODS AND MATERIALS
Table 1 lists the 18 species analysed in the current work
together with their origin.
Genome size estimation
Two methods were used to estimate genome size in
Nicotiana species: flow cytometry and Feulgen microdensi-
tometry. Both methods have been shown to produce com-
parable results (Dolez ˇel et al., 1998).
Feulgen microdensitometry using a Vickers M85a micro-
densitometer followed the methods described in Hanson
et al. (2001), and flow cytometry was conducted using a
Partec CyFlow or PAII as described in Hanson et al.
(2005). The calibration standard and method used for
each species are listed in Table 1. Expected genome sizes
of the polyploids were determined by adding the genome
sizes of the putative diploid progenitor species.
GISH was carried out as described in Clarkson et al.
(2005). Briefly, slides were pretreated with RNase A
(100 mg mL–1, 1 h) and pepsin (0.25 mg mL–1, 5 min), fol-
lowed by denaturation in 70 % formamide in 2? SSC
(0.3 M sodium chloride, 0.03 M sodium citrate) at 708C
for 2 min. The hybridization mixture included 8 mg mL–1
digoxigenin-labelled N. sylvestris DNA and 8 mg mL–1
biotin-labelled N. obtusifolia DNA. In situ hybridization
was carried out overnight at 37 8C. Post-hybridization
washes included formamide [20% (v/v) in 0.1? SSC,
42 8C] giving an estimated hybridization stringency of
80–85 %. Sites of probe hybridization were detected with
Biochemicals) (20 mg mL–1) and Cy3-conjugated avidin
(Amersham Biosciences;5 mg mL–1). Chromosomes
phenylindole; 2 mg mL–1in 4? SSC,) and mounted in
Vectashield medium (Vector Laboratories). Metaphases
were photographed on a Leica DMRA2 epifluorescence
microscope with an Orca ER camera. Images were pro-
Genome size estimates in Nicotiana
Table 1 lists genome size estimates obtained for the species
analysed. Figure 2 shows the genome size of the diploid
species considered to be most closely related to the
species that gave rise to the polyploids, together with the
expected versus the observed genome size estimates for
these polyploids. Genome downsizing was observed in
four of the polyploids studied and genome size increases
were observed in the remaining five polyploids.
Comparisons between genome size estimates obtained in
the present work for 16 species with those reported by
Narayan (1987) are shown diagrammatically in Fig. 3. For
11 species Narayan’s estimates were larger than ours
whereas in the remaining five species our estimates were
greater than Narayan’s.
GISH in section Repandae
When GISH was applied to chromosome preparations of
the polyploid species N. nudicaulis and N. nesophila (both
in section Repandae) the labelling patterns were quite
different (Fig. 4). In N. nudicaulis both probes (i.e.
genomic DNA from N. sylvestris and N. obtusifolia)
hybridized weakly across all chromosomes, although
some chromosomes were more strongly labelled with
N. sylvestris probe (green signal) and others were more
strongly labelled with the N. obtusifolia probe (red
signal). Given the amount of cross-hybridization it was
not possible to distinguish the genomic origin of each
chromosome in N. nudicaulis.
In contrast, hybridization of both probes to chromosomes
of N. nesophila was weak, with only scattered signal
FIG. 3. Comparison between 1C DNA amounts reported by Narayan
(1987) (closed triangles) and those given in Table 1 of this paper (open
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana809
detected from each probe. The only exception to this was
the strong yellow hybridization signals corresponding to
the location of ribosomal DNA loci.
Comparisons with previous genome size estimates
The most extensive survey of genome sizes in species of
Nicotiana prior to the present one was that of Narayan
(1987) who estimated DNA amounts in 51 species.
However, as illustrated in Fig. 3, there are considerable dis-
crepancies between some of his values and those estimated
here (Table 1). The most notable are those for N. rustica for
which Narayan’s estimate is nearly one-third larger than
ours (1C ¼ 7.2 versus 5.3 pg), and N. stocktonii for which
our estimate is nearly double that of Narayan’s (1C ¼ 5.0
versus 2.8 pg). Further discrepancies are noted between
workers. For example, whereas Galbraith et al. (1983)
and we (Table 1) reported 1C values of 2.1 and 2.2 pg,
respectively, for N. glutinosa, Narayan gave an estimate
of 1C ¼ 3.9 pg. The reasons for these discrepancies
remain unknown, but the observation that the differences
are not always in the same direction (i.e. sometimes
Narayan’s estimates are larger or smaller than ours and pre-
vious workers) suggests that there is not a simple, methodo-
discrepancies may be due to taxonomic issues (e.g. incor-
those published byother
Evolution of genome size in Nicotiana polyploids
It is noted that the observations and discussions presented
below assume that (a) the diploid species used are indeed
closely related to the actual progenitors that gave rise to
the polyploids, and that (b) since polyploid formation the
genome sizes of these diploids have remained largely
unchanged. Any violation of these two assumptions will
affect interpretation of the results, but at present it is the
best approximation available. Choice of diploid progenitor
is based on extensive molecular and morphological ana-
lyses of the majority of species in the genus and is thus
as comprehensive as possible. The possibility that the incor-
rect species have been selected is low but cannot be ruled
out completely, particularly for the older polyploids. The
second assumption is impossible to confirm as it requires
estimating genome sizes in the diploid progenitor and poly-
ploid species at the time of polyploidization – thousands to
millions of years ago (depending on the polyploid). With
these caveats, the following results and discussion on the
evolution of genome size in polyploids of Nicotiana are
Polyploids ,200 000 years old. Three independently pro-
duced allotetraploids of Nicotiana (all 2n ¼ 4x ¼ 48),
N. tabacum, N. rustica and N. arentsii, are estimated to
be approx. 200 000 years old or less. Based on flower and
chromosome morphology (Goodspeed, 1954) and plastid
and nuclear DNA sequence data (Aoki and Ito, 2000;
Chase et al., 2003) the parental genomes of these poly-
ploids are considered to be derived from ancestors of the
extant species shown in Fig. 2A–C. All three polyploids
appear to have undergone a limited amount of genome
downsizing, losing between 3.7 % and 5.4 % over approx.
200 000 years. These results agree with the lower
numbers of certain repeats reported in these polyploids
compared with their diploid progenitors. For example, in
N. tabacum there are lower numbers of several repeat
sequences, including NTRS, A1/A2 (Lim et al., 2004), a
pararetroviral repeat sequence NtoEPRV (Gregor et al.,
2004) and various retrotransposons (Melayah et al., 2004;
Petit et al., 2007), than in the diploids. In N. rustica, an
examination of satellite repeats showed they too differed
in abundance and distribution relative to the diploid pro-
genitor species (Lim et al., 2005). One repeat (NUNSSP)
was similar in organization and copy number to the
diploid progenitors, whereas a comparison of a second
repeat (NPAMBO) revealed minor changes in its chromoso-
mal distribution but copy number was reduced by at least
10-fold compared with N. paniculata (the maternal donor
species used for comparison). Such sequence elimination
probably occurred since N. rustica evolved and could
have contributed to the observed 2–5% reduction in DNA
amount (Fig. 2B). Alternatively these sequences may
have increased in the diploids after polyploid formation.
In all three polyploids the number of 35S rDNA loci is
additive, yet within this framework considerable sequence
elimination of individual copies has taken place with only
30%, 50 % and 80% of the expected copy numbers
(based on estimates from the diploid species) observed in
N. tabacum, N. arentsii and N. rustica, respectively. In
N. tabacum and N. rustica, most of the remaining repeats
from the maternal genome donor have been replaced, via
gene conversion, with those originating from the paternal
genome donor (Kovarik et al., 2004, 2008). Similar obser-
vations have been reported for N. arentsii, although here the
FIG. 4. GISH to (A) Nicotiana nudicaulis and (B) N. nesophila root-tip
metaphase probed with N. sylvestris genomic DNA (digoxigenin labelled,
FITC detected, yellow/green) and N. obtusifolia (biotin labelled, Cy3
detected, red). Chromosomes were counterstained with DAPI (blue).
Scale bar ¼ 10 mm.
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana810
direction of homogenization is different, with the 35S
rDNA repeats being homogenized to the maternal repeat
type (Kovarik et al., 2004, 2008).
of theseyoung Nicotiana polyploids. In all three, the number
diploid progenitors (i.e. additive), with no evidence for
sequence loss or homogenization (Fulnecek et al., 2002).
Further, the copy number of a satellite repeat isolated from
N. paniculata (NPAMBE) was greater in N. rustica, although
the numberof extra copies varied 1.7-fold between the seven
accessions analysed (Lim et al., 2005).
Polyploids approx. 1 million years old. The two polyploid
species comprising section Polydicliae, N. quadrivalvis and
N. clevelandii, are estimated to have formed approx. 1 Myr
ago. Sequencing of both nuclear and plastid DNA indicates
that they most likely arose from two different polyploidiza-
of N. obtusifolia (section Trigonophyllae) as the maternal
genome donor and a progenitor of the lineage that later
genome donor (Chase et al., 2003; Clarkson et al., 2004;
Knapp et al., 2004; Qu et al., 2004). Analysis of genome
size shows that in contrast to the younger polyploids dis-
cussed above, these two polyploids have undergone an
increase in genome size. In N. clevelandii, genome upsizing
is small – approx. 2.5% – but in N. quadrivalvis it is more
substantial (þ7.5 %; Fig. 2D). NB These changes are based
on using the genome size of N. attenuata as the species
most closely related to the paternal genome donor of
tor of N. attenuata or an ancestor of all species in section
Petunioides, Chase et al., 2003), an additional estimate of the
observed versus expected genome size in Polydicliae was
the paternal genome donor (mean 1C ¼ 2.57 pg; I. J. Leitch;
L. Hanson, unpubl. res.). Using this approach differences in
genome size were still detected for N. clevelandii and
N. quadrivalvis but were smaller than just using the value for
N. attenuata (þ0.5% and þ6%, respectively).
Currently there is little information on specific molecular
changes that have accompanied evolution of species in
section Polydicliae, although a reduction in genome size
might be expected based on the chromosomal distribution
of 35S and 5S rDNA sites using FISH. Kovarik et al.
(2008) showed a loss of loci; both polyploids had just
three 35S and one 5S loci compared with the expected
five 35S and two 5S loci based on the numbers of loci
observed in N. attenuata and N. obtusifolia (Kovarik
et al., 2008). In addition, Wu et al. (2006) analysed
low-copy genes encoding a family of trypsin-proteinase
inhibitors. From cDNA, intron and promoter sequence
analysis and Southern blotting, they deduced that only the
maternally inherited genes of N. obtusifolia were retained
in both N. clevelandii and N. quadrivalvis whereas those
of N. attenuata were deleted. Thus there is apparent
incongruence between the sequence data showing mostly
uniparental eliminations and the increased genome sizes.
An explanation may stem from the GISH results on
N. quadrivalvis by Lim et al. (2007) showing (a) interge-
nomic mixing of DNA between the two parental genomes
and (b) the invasion of N. attenuata subtelomeric repeat
sequences onto N. obtusifolia chromosomes followed by
their replacement. Adding the genome size data to these
observations suggests that genome evolution in these poly-
ploids has been accompanied by increases in the number of
existing repeats. Perhaps increase in genome size arises
from copy and paste mechanisms that also blur the distinc-
tion between the two parental genomes, as revealed by
GISH (Lim et al., 2007). Further work is needed to charac-
terize the nature and number of a representative sample of
repeats in these polyploids.
Intergenomic sequence invasions involving both rDNA
(Wendel et al., 1995) and certain classes of transposable
elements (Zhao et al., 1998) have been observed in
Gossypium polyploids that are estimated to be between 1
and 2 Myr old and thus similar in age to species in
section Polydicliae. However, whether these rates of inter-
genomic sequence invasions in polyploid nuclei are ubiqui-
tous or widespread in other polyploid groups of these ages
needs to be determined.
Polyploids approx. 4.5 million years old. Section Repandae
comprises four polyploid
N. repanda, N. stocktonii and N. nesophila (Knapp et al.,
2004). Based on DNA sequence data from both the internal
transcribed spacer of nuclear rDNA (Chase et al., 2003) and
plastid DNA (Clarkson et al., 2004) the group has been
shown to be monophyletic with N. nudicaulis sister to
and distinct (both morphologically and genetically) from
the remaining three species (which differ minimally).
Clarkson et al. (2005) suggested that the original allopoly-
ploidization event occurred approx. 4.5 Myr ago. This was
followed by subsequent speciation with N. nudicaulis diver-
ging from the rest approx. 2–3 Myr ago and N. stocktonii
and N. nesophila diverging from N. repanda more recently,
approx. 1 Myr ago. Sequence data from glutamine synthase
(Clarkson et al., 2005) and plastid loci (Clarkson et al.,
2004) indicate that the maternal genome donor of these
four polyploids was an ancestor of section Sylvestres,
which today comprises a single species N. sylvestris
(1C ¼ 2.7 pg). The paternal genome donor is an ancestor
of section Trigonophyllae, which also now comprises a
single extant species N. obtusifolia (1C ¼ 1.5 pg). If
genome size evolution in the four polyploids were additive
then one would expect each to have a 1C value of 4.2 pg.
However, this is not the case (Table 1, Fig. 2). Instead,
the following were observed: (a) genome decreases in
N. nudicaulis (1C ¼ 3.6 pg) with a loss of approx. 14.3%
of DNA comparedwith
(b) genome upsizing of 28.6 % in N. repanda (1C ¼
5.4pg) and 19.1 % in N. nesophila and N. stocktonii (both
with 1C ¼ 5.0 pg).
There are considerable differences in the GISH-labelling
patterns between N. nudicaulis (Fig. 4A) and N. nesophila
(Fig. 4B). GISH to N. nudicaulis gives substantial labelling
species, N. nudicaulis,
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana811
of the chromosomes, with some chromosomes that predo-
minantly label with N. sylvestris genomic DNA and
others predominantly with N. obtusifolia genomic DNA.
However, due to cross hybridization of the probes, it is
not possible to distinguish the parental origin of most
chromosomes of the complement. In contrast, GISH to
N. nesophila generates little signal at all, with only rDNA
(large yellow signal in Fig. 4B) and some minor signals
from both GISH probes. Lim et al. (2007) described the
loss of signal as ‘near-complete genome turnover’ (see
also Grover et al., 2007a).
Comparing GISH signals with genome size data is infor-
mative. The presence of some GISH signal to N. nudicaulis
is associated with genome downsizing, whereas the near
absence of GISH signal to N. nesophila is associated with
genome size increases. Although mobility of repeats and
homogenization mechanisms would act to reduce GISH dis-
crimination between the two parental chromosome sets, it
would not almost completely eliminate GISH signal, as
seen in N. nesophila. Genome downsizing is thought to
involve illegitimate recombination leading to the pro-
duction of small indels and unequal intrastrand homologous
recombination in, for example, long terminal repeat retroe-
lements (e.g. Devos et al., 2002; Vitte and Panaud, 2003;
Bennetzen et al., 2005). If these mechanisms are occurring
in N. nudicaulis then the overall similarity of the repeat
elements must be retained sufficiently to enable GISH to
work partially. In contrast, near absence of GISH signal
on N. nesophila is associated with genome upsizing.
Mechanisms responsible for this phenomenon include
amplification, transposition and insertion of retroelements
(Vitte and Bennetzen, 2006) and evolution and amplifica-
tion of satellite repeats (Lim et al., 2000, 2006a).
Presumably these mechanisms, including evolution of the
new satellite repeat NNE in N. nesophila, are occurring
so rapidly that almost the entire repetitive portion of the
genome has ‘turned over’, and little of the original character
of the chromosomes remains (Lim et al., 2007). Thus, these
results suggest that genome downsizing leads to a less dra-
matic alteration of genome characteristics than genome
upsizing. It is likely that evolution and amplification of
new sequences in association with genome upsizing
replace many of the original sequences that were in the
ancestral polyploid genome.
Genomic responses to polyploidy in Nicotiana are complex,
variable and determined by many factors, with age and
genomic similarity of the parental genome donors poten-
tially playing a role. Age is important in determining the
extent of DNA sequence divergence encountered in poly-
ploids, with genome turnover becoming extensive after
approx. 4.5 Myr (Lim et al., 2007). Genomic relatedness
of the parental genome donors may also be important in
determining the extent of genome evolution in the poly-
ploid genome. In Nicotiana the only polyploid shown to
have undergone unequivocal intergenomic translocations
is N. tabacum, which combines genomes from phylogeneti-
cally widely separated sections (Fig. 1; Kenton et al., 1993;
Lim et al., 2004). The next most highly diverged genomes
brought together are found in section Repandae, although
genomic turnover and homogenization that has taken
place since the four polyploids formed prevents GISH
from effectively identifying any ancestral intergenomic
translocations. No intergenomic translocations have been
found in any of the other polyploids examined (Chase
et al., 2003; Lim et al., 2004, 2007). These observations
are similar to those of Song et al. (1995), who noted that,
in synthetic polyploids of Brassica, the most extensive
genomic changes occurred in polyploids with the most
widely diverged parental genomes.
In terms of genome size evolution, no trends with
increasing age of the polyploids or increasing distance
between diploid progenitors of the polyploids were discern-
able, with four Nicotiana polyploids showing decreases and
five showing increases. Even different polyploids originat-
ing from the same parental genome donors (i.e. section
Repandae) responded differently
(N. nudicaulis) exhibiting decreases and the other three
(N. nesophila, N. stocktonii and N. repanda) showing
increases. The only noticeable trend was an increase in
the extent of DNA amount change with increasing age
of the polyploids. Thus, whereas ‘young’ polyploids
(,200 000 years old) showed only small losses of DNA
(2–5 %), after approx. 4.5 Myr of evolution DNA amount
changes ranged from 14 % to 29 % (Fig. 2). The potential
relationship between genome size increases and turnover
(replacement of ancestral repeats with new repeats over
time) and genome decreases with maintenance of ancestral
repeats will need to be explored.
Studying this relationship in other systems may, however,
be difficult because the only other reliable examples of
genome increases in naturally occurring polyploids are
found in a few species of Hordeum (Jakob et al., 2004).
The scarcity of genome size increases in polyploids in
general suggests strong selection against this process.
Perhaps the reason is that a newly formed polyploid
already has a large genome compared with its diploid par-
ental competitors so selection favours early polyploids
which have undergone genome size decreases rather than
increases. Nevertheless, the identification of genome size
increases in five Nicotiana polyploids suggests that here
either the additional DNA does confer some competitive
advantage or, perhaps more likely, it has no function but
there is no strong selection against it.
with one species
We thank NERC and the Grant Agency of the Czech
Republic (521/07/0116) for support.
Aoki S, Ito M. 2000. Molecular phylogeny of Nicotiana (Solanaceae)
based on the nucleotide sequence of the matK gene. Plant Biology
Bennetzen J, Ma J, Devos KM.2005. Mechanisms of recent genome size
variation in flowering plants. Annals of Botany 95: 127–132.
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana 812
Chase MW, Knapp S, Cox AV, Clarkson JJ, Butsko Y, Joseph J, et al.
2003. Molecular systematics, GISH and the origin of hybrid taxa in
Nicotiana (Solanaceae). Annals of Botany 92: 107–127.
Clarkson JJ, Knapp S, Garcia VF, Olmstead RG, Leitch AR,
Molecular Phylogenetics and Evolution 33: 75–90.
Clarkson JJ, Lim KY, Kovarik A, Chase MW, Knapp S, Leitch AR.
2005. Long-term genome diploidization in allopolyploid Nicotiana
section Repandae (Solanaceae). New Phytologist 168: 241–252.
DadejovaM, LimKY, Souckova-Skalicka
Grandbastien MA, Leitch A, et al.. 2007. Transcription activity of
rRNA genes correlates with a tendency towards intergenomic hom-
ogenization in Nicotiana allotetraploids. New Phytologist 174:
Devos KM, Brown JKM, Bennetzen JL. 2002. Genome size reduction
through illegitimate recombination counteracts genome expansion in
Arabidopsis. Genome Research 12: 1075–1079.
Dhillon SS, Wernsman EA, Miksche JP. 1983. Evaluation of nuclear
DNA content and heterochromatin changes in anther-derived diha-
ploids of tobacco (Nicotiana tabacum) cv. Coker 139. Canadian
Journal of Genetics and Cytology 25: 169–173.
Dolez ˇel J, Greilhuber J, Lucretti S, Meister A, Lysak MA, Nardi L,
et al. 1998. Plant genome size estimation by flow cytometry: inter-
laboratory comparison. Annals of Botany 82 (Suppl. A): 17–26.
Fulnecek J, Lim KY, Leitch AR, Kovarik A, Matyasek R. 2002.
Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana
tabacum and its putative parental species. Heredity 88: 19–25.
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP,
Firoozabady E. 1983. Rapid flow cytometric analysis of the cell
cycle in intact plant tissues. Science 220: 1049–1051.
Goodspeed TH. 1954. The genus Nicotiana. Waltham, MA: Cronica
Gregor W, Mette MF, Staginnus C, Matzke MA, Matzke AJM. 2004.
A distinct endogenous pararetrovirus family in Nicotiana tomentosi-
formis, a diploid progenitor of polyploid tobacco. Plant Physiology
Grover C, Hawkins JS, Wendel JF. 2007a. Tobacco genomes quickly go
up in smoke. New Phytologist 175: 599–602.
Grover CE, Kim H, Wing RA, Paterson AH, Wendel JF. 2007b.
Microcolinearity and genome evolution in the AdhA region of
diploid and polyploid cotton (Gossypium). The Plant Journal 50:
Hanson L, McMahon KA, Johnson MAT, Bennett MD. 2001. First
nuclear DNA C-values for 25 angiosperm families. Annals of
Botany 87: 251–258.
Hanson L, Boyd A, Johnson MAT, Bennett MD. 2005. First nuclear
DNA C-values for 18 eudicot families. Annals of Botany 96:
Ingle J, Timmis JN, Sinclair J. 1975. Relationship between satellite
deoxyribonucleic acid, ribosomal ribonucleic acid gene redundancy,
and genome size in plants. Plant Physiology 55: 496–501.
Jakob SS, Meister A, Blattner FR. 2004. Considerable genome size vari-
ation of Hordeum species (Poaceae) is linked to phylogeny, life form,
ecology, and speciation rates. Molecular Biology and Evolution 21:
Kashkush K, Feldman M, Levy AA. 2002. Gene loss, silencing and acti-
vation in a newly synthesized wheat allotetraploid. Genetics 160:
Kenton A,Parokonny AS,Gleba
Characterization of the Nicotiana tabacum L. genome by molecular
cytogenetics. Molecular & General Genetics 240: 159–169.
Knapp S, Chase MW, Clarkson JJ. 2004. Nomenclatural changes and a
new sectional classification in Nicotiana (Solanaceae). Taxon 53:
Kovarik A, Fajkus J, Koukalova B, Bezdek M. 1996. Species-specific
evolution of telomeric and rDNA repeats in the tobacco composite
genome. Theoretical and Applied Genetics 92: 1108–1111.
Kovarik A, Matyasek R, Lim KY, Skalicka K, Koukalova B, Knapp S,
2004. Concerted evolution of 18-5.8-26S rDNA repeats in
Nicotiana allotetraploids. Biological Journal of the Linnean Society
YY, BennettMD. 1993.
Kovarik A, Dadejova M, Lim KY, Chase MW, Clarkson JJ, Knapp S,
et al. 2008. Evolution of rDNA in Nicotiana allopolyploids: a poten-
tial link between rDNA homogenization and epigenetics. Annals of
Botany 101: 815–823.
Leitch IJ, Bennett MD. 2004. Genome downsizing in polyploid plants.
Biological Journal of the Linnean Society 82: 651–663.
Levy AA, Feldman M. 2004. Genetic and epigenetic reprogramming of
the wheat genome upon allopolyploidization. Biological Journal of
the Linnean Society 82: 607–613.
Lim KY, Matyasek R, Lichtenstein CP, Leitch AR. 2000. Molecular
cytogenetic analyses and phylogenetic studies in the Nicotiana
section Tomentosae. Chromosoma 109: 245–258.
Lim KY, Matyasek R, Kovarik A, Leitch AR. 2004. Genome evolution
in allotetraploid Nicotiana. Biological Journal of the Linnean Society
Lim KY, Matyasek R, Kovarik A, Fulnecek J, Leitch AR. 2005.
Molecular cytogenetics and tandem repeat sequence evolution in the
allopolyploid Nicotiana rustica compared with diploid progenitors
N. paniculata and N. undulata. Cytogenetic and Genome Research
Lim KY, Kovarik A, Matyasek R, Chase MW, Knapp S, McCarthy E,
et al. 2006a. Comparative genomics and repetitive sequence diver-
gence in the species of diploid Nicotiana section Alatae. The Plant
Journal 48: 907–919.
Lim KY, Souckova-Skalicka K, Sarasan V, Clarkson JJ, Chase MW,
Kovarik A, et al. 2006b. A genetic appraisal of a new synthetic
Nicotiana tabacum (Solanaceae) and the Kostoff synthetic tobacco.
American Journal of Botany 93: 875–883.
Lim KY, Kovarik A, Matyasek R, Chase MW, Clarkson J,
Grandbastien MA, et al. 2007. Sequence of events leading to near-
complete genome turnover in allopolyploid Nicotiana within five
million years. New Phytologist 175: 756–763.
Matyasek R, Lim KY, Kovarik A, Leitch AR. 2003. Ribosomal DNA
evolution and gene conversion in Nicotiana rustica. Heredity 91:
Matzke M, Gregor W, Mette MF, Aufsatz W, Kanno T, Jakowitsch J,
et al. 2004. Endogenous pararetroviruses of allotetraploid Nicotiana
N. tomentosiformis. Biological Journal of the Linnean Society 82:
Melayah D, Lim KY, Bonnivard E, Chalhoub B, De Borne FD,
Mhiri C, et al. 2004. Distribution of the Tnt1 retrotransposon
family in the amphidiploid tobacco (Nicotiana tabacum) and its
wild Nicotiana relatives. Biological Journal of the Linnean Society
Messing J, Bharti AK, Karlowski WM, Gundlach H, Kim HR, Yu Y,
2004. Sequence composition and genome organization of
maize. Proceedings of the National Academy of Sciences of the
USA 101: 14349–14354.
Narayan RKJ. 1987. Nuclear DNA changes, genome differentiation and
Evolution 157: 161–180.
Narayan RKJ, Rees H. 1974. Nuclear DNA, heterochromatin and phylo-
geny of Nicotiana amphidiploids. Chromosoma 47: 75–83.
Ozkan H, Levy AA, Feldman M. 2001. Allopolyploidy-induced rapid
genome evolution in the wheat (Aegilops-Triticum) group. The
Plant Cell 13: 1735–1747.
Ozkan H, Tuna M, Arumuganathan K. 2003. Nonadditive changes in
(Aegilops-Triticum) group. Journal of Heredity 94: 260–264.
Petit M, Lim K, Julio E, Poncet C, Dorlhac de Borne Fo, Kovarik A,
et al. 2007. Differential impact of retrotransposon populations on the
genome of allotetraploid tobacco (Nicotiana tabacum). Molecular
Genetics and Genomics 278: 1–15.
Qu N, Schittko U, Baldwin IT. 2004. Consistency of Nicotiana attenua-
ta’s herbivore- and jasmonate-induced transcriptional responses in the
allotetraploid species Nicotiana quadrivalvis and Nicotiana clevelan-
dii. Plant Physiology 135: 539–548.
Skalicka K, Lim KY, Matyasek R, Koukalova B, Leitch AR,
Kovarik A. 2003. Rapid evolution of parental rDNA in a synthetic
tobacco allotetraploid line. American Journal of Botany 90: 988–996.
Skalicka K, Lim KY, Matyasek R, Matzke M, Leitch AR, Kovarik A.
2005. Preferential elimination of repeated DNA sequences from the
progenitors, N. sylvestrisand
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana813
paternal, Nicotiana tomentosiformis genome donor of a synthetic,
allotetraploid tobacco. New Phytologist 166: 291–303.
Song KM, Lu P, Tang KL, Osborn TC. 1995. Rapid genome change in
synthetic polyploids of Brassica and its implications for polyploid
evolution. Proceedings of the National Academy of Sciences of the
USA 92: 7719–7723.
Vitte C, Bennetzen JL. 2006. Analysis of retrotransposon structural diver-
sity uncovers properties and propensities in angiosperm genome evol-
ution. Proceedings of the National Academy of Sciences of the USA
Vitte C, Panaud O. 2003. Formation of solo-LTRs through unequal hom-
ologous recombination counterbalances amplifications of LTR retro-
transposons in rice Oryza sativa L. Molecular Biology and
Evolution 20: 528–540.
Wang XY, Shi XL, Hao BL, Ge S, Luo JC. 2005. Duplication and DNA
segmental loss in the rice genome: implications for diploidization.
New Phytologist 165: 937–946.
Wendel JF, Schnabel A, Seelanan T. 1995. Bidirectional interlocus con-
certed evolution following allopolyploid speciation in cotton
(Gossypium). Proceedings of the National Academy of Sciences of
the USA 92: 280–284.
Wu J, Hettenhausen C, Baldwin IT. 2006. Evolution of protein inhibitor
defenses in North American allopolyploid species of Nicotiana.
Planta 224: 750–760.
Zhao XP, Si Y, Hanson RE, Crane CF, Price HJ, Stelly DM, et al. 1998.
Dispersed repetitive DNA has spread to new genomes since polyploid
formation in cotton. Genome Research 8: 479–492.
Leitch et al. — Genome Size Evolution in Polyploid Species of Nicotiana814